Abstract: Changes in environmental conditions can affect gene expression at many levels. In general, alterations in transcription have been the most widely explored consequences of environmental perturbations, but changes at the level of the translation of specific mRNAs can also have profound effects on a cell's ability to respond to environmental stresses and to damage caused by toxins or other external agents. In addition, execution of critical genetic programs that determine a cell's fate and function - including many developmental processes - frequently begins at the translational level. Disregulation of the initiation phase of protein synthesis plays a role in the etiology of a variety of human diseases, from cancers to neurodegenerative disorders. The selection of the start codon in an mRNA by the translational machinery is a critical phase of gene expression because it determines both the N-terminus of the protein that will be produced and the reading frame of decoding. There is considerable circumstantial evidence that the fidelity of start codon recognition can be affected by environmental conditions - for example, exposure to stressors and toxins or changes in the levels of specific nutrients - and that these changes alter gene expression in ways that affect the physiology of the cell. Changes in the fidelity of start codon recognition could result in production of protein isoforms with different N-termini created by translation initiation from alternative start codons in the 5'-leaders of mRNAs. It could also change expression levels of proteins by modulating translation of regulatory upstream open reading frames in mRNAs. These alterations in gene expression could be harmful to the cell, if unprogramed, or beneficial, if they are part of a posttranscriptional gene regulatory mechanism that allows the cell to respond to its altered environment or internal or external signals. Using an established, high-throughput dual-luciferase assay for measuring the fidelity of start codon recognition we will: 1) Test the hypothesis that external conditions, signals and internal cues can modulate the fidelity of start codon recognition to alter gene expression at the translational level; 2) Determine the effects of forcing changes in the fidelity of start codon recognition on cell physiology and gene expression. These studies have the potential to uncover a completely new mode of post-transcriptional gene regulation. They will also provide important insights into how environmental conditions affect gene expression at the translational level, as well as the consequences of these changes for cell physiology.